The internalization of receptors and ligands via endocytosis requires dramatic reorganization of the cell membrane, coordinated by extracellular and intracellular signals. Although membrane reorganization is a feature of both clathrin- and caveolae-mediated endocytosis, the mechanism by which it is achieved - and how it is coordinated with specific cues - remains unclear. One set of candidates involved in membrane reorganization prior to caveolae-mediated endocytosis are the annexins, a family of proteins which translocate to the membrane in a calcium-dependent manner, where they associate with lipid microdomains and may form two-dimensional scaffolds [Gerke, Nat. Rev. Mol. Cell Biol., 2005]. This behavior suggests a possible role for annexins in organizing and maintaining caveolae, regions of the membrane where lipid microdomains and GPI-anchored proteins in the outer leaflet are believed to cluster prior to internalization [Pelkmans, Traffic, 2002]. While certain annexins bind cholesterol in a calcium-independent manner and localize to the cytoplasmic side of caveolae [Mayran, EMBO Journal, 2003; Lisanti, J. Cell Biol., 1994], it remains an open question as to how the calcium-independent interactions of annexins with cholesterol are balanced with their calcium-dependent interactions with other membrane components, and if annexins themselves play an active role in coordinating the clustering of lipid microdomains and GPI-anchored proteins into caveolae from the cytoplasmic side of the membrane. This research proposes to address this question by reconstructing elements of membrane- annexin interactions in vitro, where the stimulating calcium signal can be spatially and temporally controlled, and the annexin response determined under biochemically well-defined conditions. This will be accomplished by encapsulating the annexin AnxA2 in lipid vesicles with controlled membrane composition (Aim 1), studying the effects of cholesterol on AnxA2 translocation (Aim 2), and investigating the effects of annexin A2 translocation and distribution on the distributions of proteins linked to the outer leaflet of the membrane (Aim 3). Accomplishing these aims will help to elucidate the mechanism through which annexins participate in caveolae-mediated endocytosis. A better mechanistic understanding of this fundamental cellular process could help in identifying new targets for treating pathologies associated with caveolae, including many lipid storage diseases [Marks, Trends Cell Biol., 2002].